Ab48789

Frozen tissue sections stored in −80 °C were thawed in RT to be fixated with 3% freshly made paraformaldehyde in TBS for 10 min in RT. Tissues were then permeabilized for 10 min in TBS+ 0.1%Triton-X100, rinsed three times in TBS for 5 min/ rinse. Blocking with 2% bovine serum albumin in TBS for 2 h was performed before the tissues were incubated with primary antibodies overnight at 4 °C. After rinsing with 3 × 5 min with TBS the tissues were incubated with the secondary antibodies donkey anti-mouse immunoglobulin G (IgG)-AlexaFluor 568 (1:500 Molecular Probes) and donkey anti-rabbit IgG-AlexaFluor 647 (1:500 Molecular Probes) for 1 h at RT in darkness. DNA was counterstained with DAPI (Molecular Probes) and slides were mounted with Prolong Gold (Molecular probes). Tissues were stained with antibodies against SPINK1 (1:50, H00006690-M01, 4D4, Novus), TFF3(1:200, HPA 035464, Sigma), SPON2 (1:100, A-10, st cruz), PGC (1:50, NBP1-91011, Novus), NPY (1:100, ab48789, Abcam), Aquaporin (1:100, ab168387, Abcam), NR4A1 (1:100, ab48789, Abcam), ACPP (1:100, Biologicals), P63(1:150, ab53039, Abcam), Vimentin (1:150, ab8069, Abcam). Fluorescence images were obtained with a Zeiss LSM 780 inverted confocal microscope, using a Plan Apochromat 20 × /NA (numerical aperture) 0.7 objective. Tiled images were acquired from optical sections of 5 micrometer.